基于Arah6的花生间接竞争ELISA检测方法的建立
A method for detecting peanut based on Ara h 6 by indirect competitive enzyme-linked immuno sorbent assay
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摘要: 建立了基于花生主要过敏原蛋白Arah6的间接竞争酶联免疫检测法,实现了对食物中花生的定量检测。利用花生过敏原Arah6纯品免疫新西兰大白兔,制得兔抗Arah6的多克隆抗体,以Arah6纯品作为包被抗原、自制抗体为一抗、辣根过氧化物酶标记的羊抗兔IgG为二抗,确定了包被抗原质量浓度为1μg/mL,一抗最佳稀释度为1:50000,酶标二抗稀释度为1:5000。此检测方法对Arah6的定量检测范围为16.5～10000ng/mL（折合成含花生的含量,约为165ng/mL～100μg/mL）,抑制方程为抑制率I=21.418lgC-6.0633（C为Arah6的质量浓度,单位ng/mL）,IC50为414.6ng/mL,相关系数R2=0.9989。该检测方法灵敏度高,检测范围广,适用于食品中花生现场快速检测。Abstract: An indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed to detect peanut in food, based on Ara h 6, a main allergen in peanut.The polyclonal antibody was prepared by immunizing New Zealand rabbits with Ara h 6.Those parameters were optimized as following.The concentrations of the coating antigen, polyclonal antibody against Ara h 6 and horseradish peroxidase conjugated goat anti-rabbit IgG were 1μg/mL, 1:50000 and 1:5000 respectively.The inhibition equation of indirect competitive ELISA was obtained as I= 21.418lgC-6.0633 (I stand for inhibition ratio and C stand for mass concentration) and the curve was linear in range of 16.5～10000ng/mL.Thus, this method has good sensitivity and wide range for the detection of Ara h 6 in peanut food.
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